Hyaluronic acid (HA) is a polysaccharide composed of a disaccharide motif comprising D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating β(1,4)- and β(1,3)-glycosidic bonds.

HA can be cross-linked by various cross-linking agents, e.g. bi- or polyfunctional epoxides, formaldehyde, gluteraldehyde and divinyl sulfone. Cross-linked HA gels using the bifunctional epoxide 1,4-butanediol diglycidyl ether (BDDE) are commercially available e.g. under the trademarks Restylane or Juvederm.
Numerous methods for chemical modification have been employed in order to functionalize and thereby impart new or modified properties to the cross-linked HA.
The chemical modifications can be realized via available —COOH groups of the HA by preactivation with carbodiimides, such as 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC). N-hydroxysuccinimide (NHS) or 1-hydroxybenzotriazole (HOBt) may also be used together with the EDC to facilitate the coupling reaction. The coupling reactions may also be achieved by the use of 1,1-carbonyldiimidazole (CDI), see e.g. WO 00/01733. These reactions can be performed in water or by adding a co-solvent, such as DMSO.
T. Pouyani & G. D. Prestwich, Bioconjugate Chemistry 5: 339-347 (1994) discloses carbodiimide-mediated coupling of hydrazides to very short oligosaccharide fragments of HA. The oligosaccharide is activated in a first step by the coupling of a hydrazide, followed by a second step where a desired substance can be reacted with the hydrazide-activated oligo-saccharide and become incorporated on the oligosaccharide. Alternatively, the hydrazide-activated oligosaccharide can in a second step be reacted with a cross-linker to obtain a cross-linked oligosaccharide.
G. D. Prestwich et al., J. Control. Release 53 (1-3): 93-103 (1998) discloses a complicated multi-step process involving carbodiimide-mediated coupling of hydrazides to HA. The HA is activated in a first step by the coupling of a hydrazide, followed by a second step where a desired substance can be reacted with the hydrazide-activated HA, and a third step of reacting this functionalized HA with an already cross-linked HA.
P. Bulpitt et al., J. Biomed. Mater. Res. 47 (2):152-169 also discloses a multi-step process involving carbodiimide-mediated coupling of hydrazides to HA. The HA is activated in a first step by the coupling of a hydrazide, followed by a second step where a desired substance can be reacted with the hydrazide-activated HA, and a third step of cross-linking this functionalized HA with a cross-linker.
WO 2007/102149 A2 also discloses a multi-step process involving carbodiimide-mediated coupling of hydrazides to HA. The HA is activated in a first step by the coupling of a hydrazide, followed by a second step of cross-linking this hydrazide-activated HA with a cross-linker. Additional components may be coupled to the hydrazide-functionalized HA in a separate step, before or after the cross-linking step.
The chemical modifications can also be realized via available —OH groups of the HA by reaction with acyl chlorides in a solvent, such as DMSO, or using anhydrides in water (pH=9), see e.g. WO 2007/033677. The —OH groups of the HA can also be functionalized with amines using cyanogen bromide as an activation reagent.
The reported methods for functionalizing cross-linked HA involve functionalizing of the HA prior to cross-linking, followed by cross-linking of the functionalized HA. This method is disadvantageous because it requires modification and purification of a large molecular weight polysaccharide, and because the cross-linking step may result in uncontrolled reaction of the cross-linking agent with the grafted molecule.
Alternatively, the reported methods for functionalizing cross-linked HA involve cross-linking the HA prior to the functionalization, followed by functionalizing of the cross-linked HA. This method is disadvantageous because of difficulties in purifying the resulting gel from by-products formed during the reaction. Moreover, the resulting gel is not homogeneous because the functionalization tends to be formed on the surface of particles.
WO 2008/031525 teaches cross-linking of HA by click chemistry. The cross-linking process involves several time-consuming steps of modifying the sensitive HA with a multiplicity of reagents to allow for the cross-linking reaction.